Thyroid stimulating hormone (TSH) is one of a family of glycoprotein hormones that consist of two non-covalently linked, dissimilar subunits, designated alpha and beta. We are investigating the cellular events that take place in the biosynthesis of TSH; specifically, we wish to understand the cellular mechanisms of post-translational processing of the alpha and beta subunits including cleavages of precursors, glycosylation, and assembly of chains. We wish to determine how these processes are regulated, how they may be interdependent, and how they may be involved in intracellular transport, packaging, and secretion of the hormone. An essential first approach to these studies is to obtain information about the primary structure of the subunits and their precursors. To accomplish this, we are: (a) determining the amino acid sequence of TSH-subunits isolated from a transplantable thyrotroph tumor developed in our laboratory, and (b) determining the primary sequences of the putative subunit precursors found by translations of thyrotroph tumor mRNA in heterologous cell-free systems. Two approaches are used for the sequences analyses: (1) radiomicrosequencing of the protein translation products, and (2) sequencing of cDNA made complementary to the subunit mRNAs. The sequencing of the cDNA will provide information about the structure of the non-coding regions of the mRNAs for the subunits. This information of the primary structures will then be used in analyses of the processes of glycosylation and post -translational cleavages of the subunit precursors. Further, we propose to analyze by electrophoresis and by electron microscopic radioautography the migration and assembly of newly synthesized subunit chains in intact thyrotrophic cells in vitro. These initial studies will then provide the basis for subsequent studies of the cellular mechanisms involved in the regulation of subunit synthesis, assembly, and secretion mediated by thyrotrophin releasing hormone and thyroid hormones.